CN107991409A - Using the efficient method closed phase chromatography and measure 12 kinds of sulfa drugs in blood plasma at the same time - Google Patents

Using the efficient method closed phase chromatography and measure 12 kinds of sulfa drugs in blood plasma at the same time Download PDF

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CN107991409A
CN107991409A CN201711212283.6A CN201711212283A CN107991409A CN 107991409 A CN107991409 A CN 107991409A CN 201711212283 A CN201711212283 A CN 201711212283A CN 107991409 A CN107991409 A CN 107991409A
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sulfa drugs
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CN107991409B (en
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张元�
李国辉
闫加庆
刘敏
沈鑫
张远远
刘佳
周海燕
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Cancer Hospital and Institute of CAMS and PUMC
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention discloses 12 kinds of the efficient of sulfa drugs in a kind of while separation determination blood plasma to close phase chromatography (Ultra Performance Convergence Chromatography, UPC2) method.This method is that acetonitrile is added in plasma sample, is centrifuged after vortex mixed, and UPC is used after supernatant liquid filtering2Method measures, using Acquity UPC2TMBEH chromatographic columns, CO2With methanol as mobile phase, gradient elution, 40 DEG C of column oven temperature, system back pressure 2100psi.The method realizes the baseline separation to 12 kinds of sulfa drugs in 6min, and minimum to be quantitatively limited to 0.5 μ g/mL, the rate of recovery is between 80.53 103.33%.This method is easy to operate, it is accurate, save the time, it is and more environmentally friendly, detected suitable for blood plasma while a variety of sulfa drugs.

Description

Using the efficient method closed phase chromatography and measure 12 kinds of sulfa drugs in blood plasma at the same time
Technical field
The present invention relates to a kind of method for measuring 12 kinds of sulfa drugs in blood plasma at the same time using phase chromatography is efficiently closed.
Background technology
Sulfa drugs (Sulfonamides, SAs) is the general name of a kind of medicine containing P-aminobenzene-sulfonamide structure. Mainly by with p-aminobenzoic acid (P-amino benzoic acid, PABA) competitive binding dihydrofolate synthetase, shadow Ring the synthesis of bacterium nucleoprotein and play bacteriostasis, which has many advantages, such as has a broad antifungal spectrum, cheap, property are stablized. Up to now, clinical practice is still relatively broad.
Earlier studies have shown that taking same dose SAs medicines, different patient's body blood concentrations have larger difference.It is conventional Blood concentration is more during experiential therapy maintains 50-100 μ g/mL.In the blood concentration scope, medicine antibacterial effect compared with To be obvious, and severe infections are faced, it is more suitable suitably to increase medicine blood concentration, and clinic usually maintains patient's blood concentration In 120-150 μ g/mL, the incidence of adverse reaction is reduced at the same time to play the antibacterial effect of higher.In order to fully study SAs's Pharmacodynamics and pharmacokinetics, the method for establishing SAs in a kind of quick, easy, high-throughout measure blood plasma are necessary.
At present, many scholars establish respectively it is a series of be directed to biological sample in SAs analysis method [Yu H, Tao Y, Chen D,et al.J Chromatogr B Analyt Technol Biomed Life Sci,2011,879(25):2653- 2662.Zhao XT,Lin QB,Song H,et al.J Agric Food Chem,2011,59(18):9800- 9805.Zhang S,Hu Q,Sun J,et al.Chin.Traditional Patent Med.,2015,37(3):542- 548.].It is more for SAs analysis methods in biological sample both at home and abroad, the most commonly used [Gao of wherein HPLC (UHPLC) detection technique R,Zhang J,He X,et al.Anal Bioanal Chem.2010,398(1):451-461.Shaaban H,Górecki T.J Sep Sci,2012,35(2):216-224.], SAs often has similar structure, no matter takes C18 columns or amino Column, separation efficiency have much room for improvement.It is one that Waters companies newly release in part of in August, 2012 that ultra high efficiency, which closes phase chromatography (UPC2), Kind isolation technics, the technology use supercritical fluid basic principle, and not only separation efficiency is high, but also with green, environmental protection, economy Etc. advantage.
The content of the invention
The object of the present invention is to provide method that is a kind of while measuring 12 kinds of sulfa drugs in blood plasma.
Method that is provided by the present invention while measuring 12 kinds of sulfa drugs in blood plasma, be using efficiently close phase chromatography into Row measure, comprises the following steps：
1) pre-treatment is carried out to plasma sample to be measured：
Suitable test plasma sample is taken, adds isometric acetonitrile, is mixed, centrifugation, takes supernatant, crosses 0.22 μm of filter Film, it is to be detected to collect filtrate；
2) it is measured using efficiently conjunction phase chromatography：
It is described efficiently close the chromatographic condition that uses of phase chromatography for：
Using Acquity UPC2BEH chromatographic columns；
Column temperature：35 DEG C -45 DEG C, system back pressure：1800psi-2700psi；
Mobile phase：Supercritical CO2With the mixed liquor of methanol；
Type of elution：Gradient elution；
Gradient elution program is：
0-5min：Supercritical CO2Volume fraction be down to 85% by 95%,
5.0-5.5min：Supercritical CO2Volume fraction be down to 80% by 85%,
5.5-6.0min：Supercritical CO2Volume fraction increase to 95% by 80%,
6.0-7.0min：Supercritical CO2Volume fraction remain 95% constant；
Column flow rate is 1.55-1.65mL/min (preferably 1.6mL/min)；
The detector that the efficiently conjunction phase chromatography uses is PDA detectors (diode array detector)；
Wherein, 12 kinds of sulfa drugs are selected from following substances：Sulfamethoxazole (Sulfamethazine, SMZ), madribon (Sulfadimethoxypyrimidine, SMM), sulfacetamide (Sulfacetamide, STD), sulfamethyldiazine (Sulfamerazine, SMR), sulphadiazine (Sulfadiazine, SDZ), sulphathiazole (Sulfathiazole, ST), sulfapryidine (Sulfapyridine, SPD), N4- sulfacetamide methyl oxazole (N4-Ac SMZ)、 N4- sulfacetamide methylpyrimidine (N4-Ac SMR)、N4- acetylsulfadiazine (N4-Ac SDZ)、N4- acetylsulfathiazole (N4-Ac ST)、N4- acetylsulfapyridine (N4-Ac SPD)。
In step 1), described to mix by the way of being vortexed, the time of vortex is 0.5-1min；The condition of the centrifugation 6~12min is centrifuged for 6000~10000rpm at 18~24 DEG C.
In step 2), the Acquity UPC2The specification of BEH chromatographic columns is 100mm × 3mm, 1.7 μm of packing material size；Institute Stating column temperature is：40℃；The system back pressure：2100psi；
Sample temperature is 25 DEG C in the chromatographic condition, and sampling volume is 2 μ L.
The testing conditions of PDA detectors described in step 2) are：
3D scanning ranges 210-600nm, 2D compensation absorbance 265nm, compensation reference 560-600nm.
It is also another object of the present invention to provide method that is a kind of while measuring the content of 12 kinds of sulfa drugs in blood plasma, It is to be measured using efficiently conjunction phase chromatography, comprises the following steps：
1) preparation of standard curve
Take a series of hybrid standard product solution of 12 kinds of sulfa drugs of concentration to add in blank plasma samples, press Prepared according to the above-mentioned method that pre-treatment is carried out to plasma sample, phase is efficiently then closed according to above-mentioned to obtained supernatant Chromatography is detected, and records 12 kinds of sulfa drugs corresponding peak area under each concentration respectively,
Using the corresponding peak area of each sulfa drugs as ordinate, using the mass concentration of corresponding sulfa drugs as horizontal stroke Coordinate, carries out linear weight recurrence, prepares the regression equation of 12 kinds of sulfa drugs respectively；
2) assay of 12 kinds of sulfa drugs described in plasma sample to be measured
Test plasma sample is prepared according to above-mentioned sample preparation methods, then to obtained supernatant according to upper The efficient phase chromatography that closes stated is detected, and records the corresponding peak area of 12 kinds of sulfa drugs respectively；By every kind of medicine The peak area value of thing substitutes into the corresponding regression equation of the medicine respectively, and 12 kinds of sulfanilamide (SN) in the test plasma sample is calculated The concentration of class medicine.
The method of the invention can be successfully used in the pharmacokinetic of sulfa drugs.
Up to now, the measure of SAs routinely still uses liquid chromatogram (or tandem mass spectrum), but such method disengaging time compared with Long, separation efficiency has much room for improvement, and organic reagent consumption is big, has much room for improvement to the friendliness of environment.In consideration of it, the present invention is built A kind of quick, efficiently, environmentally friendly new separation method, the analysis measure for SAs in biological sample are found.The method established Mainly there is some following advantage：(1) disengaging time is short, and the separation of 12 kinds of SAs can be achieved within 6 minutes；(2) separation efficiency Height, total material can reach baseline separation；(3) clean environment firendly, effectively reduces the usage amount of organic reagent.
Brief description of the drawings
Fig. 1 is 12 kinds of sulfanilamide (SN) chromatograms, (A)：Blank plasma；(B)：+ 12 kinds of sulfa drugs of blank plasma.
Embodiment
The method of the present invention is illustrated below by specific embodiment, but the present invention is not limited thereto, it is all at this All any modification, equivalent and improvement done within the spirit and principle of invention etc., should be included in the protection model of the present invention Within enclosing.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Embodiment 1,
1 materials and methods
1.1 instruments and reagent
Acquity UPC2(Waters, US), equipped with Waters PDA detectors；Chromatographic column (is purchased from Waters,Milford,MA)：(1)Waters ACQUITY UPC2BEH(1.7μm,3×100mm I.D.),(2)Waters Acquity UPC2BEH 2-EP(1.7μm,3×100mm I.D.),(3)Acquity UPC2CSHFluoro-Phenyl(1.7μ m,2.1×100mm I.D.)and(4)Acquity UPC2HSS C18SB(1.8μm,3×100mm I.D.)。TOLEDO AL204 types electronic balance (METTLER companies of Switzerland).PURELAB Ultra MK2 type ultra-pure waters instrument is purchased from ELGA companies of Britain.
SAs reference substances：Sulfamethoxazole (Sulfamethazine, SMZ, ＞ 99%, w/w), bensulfa Pyrimidine (Sulfadimethoxypyrimidine, SMM, ＞ 99%, w/w), sulfacetamide (Sulfacetamide, STD, ＞ 99%, w/w), sulfamethyldiazine (Sulfamerazine, SMR, ＞ 99%, w/w), sulphadiazine (Sulfadiazine, SDZ, ＞ 99%, w/w), sulphathiazole (Sulfathiazole, ST, ＞ 99%, w/w), sulfapryidine (Sulfapyridine, SPD, ＞ 99%, w/w), it is purchased from German Dr.Ehrenstorfer companies.N4- sulfacetamide methyl oxazole (N4- Ac SMZ, ＞ 95%, w/w), N4- sulfacetamide methylpyrimidine (N4- Ac SMR, ＞ 95%, w/w), N4- acetylsulfadiazine (N4- Ac SDZ, ＞ 95%, w/w), N4- acetylsulfathiazole (N4- Ac ST, ＞ 95%, w/w), N4- acetylsulfapyridine (N4- Ac SPD, ＞ 95%, w/w), it is purchased from Canadian Toronto Research Chemicals companies.Methanol, ethanol and isopropanol (Germany Merck companies, chromatographically pure), ammonium formate and ammonium acetate (Agilent companies of the U.S., chromatographically pure).CO2(food-grade) is purchased from Beijing Lv Yang Big Dippers Science and Technology Ltd., use for laboratory water are ultra-pure water.
The preparation of 1.2 standard solution
Precision weighs 12 kinds of sulfanilamide (SN) and each 0.01g of metabolin reference substance (being accurate to 0.0001g), with methanol dissolving and constant volume Into 100mL volumetric flasks, 100 μ g/mL hybrid standard stock solutions of each control are made.In -20 DEG C of lucifuge stored frozens, according to need Will, the standard working solution with methanol dilution into different quality concentration, 4 DEG C of stored protected from light.
1.3 chromatographic condition
Chromatographic condition：Using Acquity UPC2BEH chromatographic columns (100mm × 3mm, 1.7 μm), column temperature：40 DEG C, the system back of the body Pressure：2100psi, 25 DEG C of sample temperature, 2 μ L of sampling volume.Mobile phase is respectively methanol (B phases) and supercritical CO2(A phases), column Flow velocity is 1.6mL/min, and eluent gradient elution program is shown in Table 1.
1 gradient elution program of table
1.4PDA condition
3D scanning ranges 210-600nm, 2D compensation absorbance 265nm, compensation reference 560-600nm.
1.5 sample pre-treatments
Precision measures 0.5mL plasma samples, puts in 2mL centrifuge tubes, adds 0.5mL acetonitriles, is vortexed and mixes 30s, at 20 DEG C 10000rpm centrifuges 10min, takes supernatant, crosses 0.22 μm of filter membrane, upper machine testing.
2 results
2.1 specificity
Blank plasma is taken, sample solution is prepared according to " 1.5 sample pre-treatments " item, it is empty according to " 1.3 chromatographic condition " sample introduction White blood plasma chromatogram occurs within the 2-6min times without endogenous impurity peaks, does not influence SAs measure, this method high-specificity.Color Spectrogram is shown in Fig. 1.
2.2 standard curves and the range of linearity
2,4,10,20,40,100 μ g/mL mixed standard solutions are prepared, respectively accurate pair for measuring above-mentioned each mass concentration According to product stock solution 0.5mL, put respectively in 2mL centrifuge tubes, add blank plasma 0.5mL, mix, make into 1,2,5,10,20,50 μ The series of samples blood plasma of g/mL, is handled by the method under " 1.5 sample pre-treatments " item, takes 2 μ L sample introductions.Calculated according to S/N >=3 To the detection limit (LOD) of this method, the quantitative limit (LOQ) of this method is calculated in S/N >=10, is sat using each sulfanilamide (SN) peak area to be vertical Mark, carries out linear weight recurrence for abscissa with the mass concentration c (μ g/mL) of each sulfanilamide (SN) material, obtains regression equation.Specifically it is shown in Table 2.
Regression equation, the range of linearity and the lower limit of quantitation of 2 12 kinds of sulfonamides of table
2.3 precision and accuracy
Blank plasma is taken, by under " standard curve and the range of linearity " item, prepares basic, normal, high 3 mass concentration plasma samples (5,50,100 μ g/mL) is 6 parts each, is operated by method under " 1.5 sample pre-treatments " item, with standard curve with batch measure, repeats to survey Determine 4d, measurement result Analysis of variance, tries to achieve the precision (RSD) of this law.As a result prompt, the withinday precision RSD of this law is 1.5%~4.1%, day to day precision RSD are 1.3%~5.2%, and accuracy is 95%~99%, are satisfied by biological sample survey Provisioning request.
2.4 stability test
Blank plasma 0.5mL is taken to put in 2mL sample cells, it is accurate respectively to add 10,80,160 μ g/mL sulfamidos pair of 0.5mL According to product stock solution, mix, the Serial plasma sample of basic, normal, high 3 mass concentrations of 5,40,80 μ g/mL is made, according to " 1.5 samples Method under product pre-treatment " item is handled, and takes 2 μ L sample introductions.Another each 6 parts of sample for matching somebody with somebody 3 mass concentrations by same procedure, its In 2 parts place 5h at room temperature after, 2 parts of multigelations 5 times, 2 parts preserve 20d under freezing conditions, are handled by same procedure Sample is stated, takes 2 μ L sample introductions of supernatant.The mass concentration of each sample is obtained by standard curve, the sample with the corrresponding quality concentration of 0h Product compare.The result shows that：Plasma sample places 5h in room temperature, and freeze thawing 5 times and -20 DEG C preserves 20d and has good stability.
2.5 extraction recovery
Blank plasma 0.5mL is taken to put in 2mL sample cells, it is accurate respectively to add the storage of 10,80,160 μ g/mL sulfamidos reference substances Standby liquid 0.5mL, mixes, and the plasma sample of basic, normal, high 3 mass concentrations of 5,40,80 μ g/mL is made, by " locating before 1.5 samples Sample is handled under reason " item, takes each 2 μ L sample introductions of solution, METHOD FOR CONTINUOUS DETERMINATION 5 times respectively.The different basic, normal, high 3 kinds of mass concentrations of SAs Extraction recovery (n=5) is shown in Table 3 respectively.
3 extraction recovery of table
Up to now, the measure of SAs routinely still uses liquid chromatogram (or tandem mass spectrum), but such method disengaging time compared with Long, separation efficiency has much room for improvement, and organic reagent consumption is big, has much room for improvement to the friendliness of environment.In consideration of it, this research is built A kind of quick, efficiently, environmentally friendly new separation method has been found, has been envisaged for the analysis measure of SAs in biological sample.The side established Method mainly has some following advantage：(1) disengaging time is short, and the separation of 12 kinds of SAs can be achieved within 6 minutes；(2) separation effect Can be high, total material can reach baseline separation；(3) clean environment firendly, effectively reduces the usage amount of organic reagent.In this method Research process, this experiment emphatically investigated influence of the different separating factors to separating effect, it is specific as follows：
Influence of 3.1 chromatographic columns to different SAs separating effects：
12 kinds of SAs in this experiment, it is similar with structure height, it is more difficult to the characteristics of separating.BEH, BEH have been investigated in experiment respectively 2-EP, CSH Fluoro-Phenyl, HSS C18SB chromatographic columns, the results show that under same experimental conditions, BEH chromatographic columns obtain Best separating effect (each medicine is to reaching baseline separation, R ＞ 1.5) was obtained, and remaining chromatographic column fails to reach to complete The baseline separation of portion's material.Eluting order is：(1)SMZ,(2)SMM,(3)N4-Ac SMZ,(4)STD,(5)SMR,(6)SPD, (7)SDZ,(8)N4-Ac SMR,(9)N4-Ac SDZ,(10)N4-Ac SPD,(11)ST,(12)N4- Ac ST (see Fig. 1).
Influence of 3.2 modifying agent to different SAs separating effects：
UPC2The most common mobile phase of system is CO2, while a small amount of organic reagent is added as modifying agent to promote to separate, Separation of the different classes of modifying agent to object can produce different influences, and methanol, ethanol and second have been investigated in this experiment respectively Separating effect of the nitrile as modifying agent.3 kinds of reagents also have the selectivity of 12 kinds of SAs larger difference, wherein separating methanol time Most short and separating effect is optimal, and whole compound separating degrees can reach more than 1.5, right when ethanol and acetonitrile are as mobile phase Part sulfanilamide (SN) separating degree has much room for improvement and disengaging time is longer.
The influence of 3.3 chromatogram column temperatures and system back pressure to different SAs separating effects：
In UPC2In systems development process, chromatogram column temperature and system back pressure are to carry out two important ginsengs of separation optimization Number, from separating mechanism, changes back pressure value or chromatogram column temperature, can significantly change supercritical CO2Density and performance so that Change its eluting power and selectivity.Column temperature (30,35,40,45,50,55 DEG C) and system pressure have been investigated in this experiment respectively (1500,1800,2100,2400,2700,3000psi) on the separated influence of determinand.
Since 30 DEG C, with the rise of temperature, compound appearance time gradually extends, and consideration is because temperature rises meeting The density of supercritical fluid is reduced, the eluting power of mobile phase is reduced, causes the retention time of the compound to extend.35 DEG C with Under, compound SMM and N4- Ac SMZ can not baseline separation, with the rise of temperature, the separation degree of two compounds increases. More than 45 DEG C, N4- Ac SMZ and STD can not baseline separation, and with the rise of temperature, both separation degrees reduce.
Since 1500psi, with the rise of back pressure, compound appearance time is gradually shortened.Consideration is because back pressure liter Height can increase the density of supercritical fluid, increase the eluting power of mobile phase, reduce the retention time of the compound.Back pressure value exists 1800psi and following, SMM and N4- Ac SMZ fail baseline separation, and as the increase of back pressure, the separating degree of two compounds are in Ascendant trend；2700psi and during the above, N4- Ac SMZ and STD can not baseline separation, and with the increase of back pressure, two compounds Separating degree it is on a declining curve.
The influence of integrated temperature and system back pressure to separating degree considers, chooses 40 DEG C of column temperature, system back pressure is that 2100psi is Optimal conditions.
Influence of 3.4 elution programs to different SAs separating effects：
This research has investigated 80% respectively, 85%, 90%, 95%CO2Isocratic elution under the conditions of four kinds, finds 80%CO2Deng Degree elution can cause whole chromatographic peaks to be eluted in 1.8 minutes, be unable to reach baseline separation, 85%CO2Isocratic elution whole thing Matter appearance, separating effect can also be unsatisfied with 3.2min, 90%CO2Isocratic elution, total material can in appearance in 9min, but To madribon and N4- sulfacetamide methyl oxazole fails to separate, 95%CO2Isocratic elution, disengaging time exceed 10min, and the flat width of peak type, it is unsatisfactory, based on the above results using gradient elution.
5 kinds of Gradient programs have been investigated in experiment respectively, finally found that elution program：0min：95%CO2；5min：85% CO2；5.5min：80%CO2；6min：95%CO2；7min：95%CO2, the baseline separation to whole test substances can be obtained (separating degree ＞ 1.5).
Influence of 3.5 chromatographic flow rates to different SAs separating effects：
This research has investigated 1.2-2.2mL/min flow velocitys to separated influence respectively.The results show that the increasing with flow velocity Add, compound appearance time significantly shortens.Comprehensive separating effect and the rate of departure consider, select 1.6mL/min flow velocitys effect most It is excellent.

Claims (9)

1. method that is a kind of while measuring 12 kinds of sulfa drugs in blood plasma, is measured using efficiently conjunction phase chromatography, including Following step：
1) pre-treatment is carried out to plasma sample to be measured：
Suitable test plasma sample is taken, adds isometric acetonitrile, is mixed, centrifugation, takes supernatant, crosses 0.22 μm of filter membrane, receives It is to be detected to collect filtrate；
2) it is measured using efficiently conjunction phase chromatography：
It is described efficiently close the chromatographic condition that uses of phase chromatography for：
Using Acquity UPC2BEH chromatographic columns；
Column temperature：35 DEG C -45 DEG C, system back pressure：1800psi-2700psi；
Mobile phase：Supercritical CO2With the mixed liquor of methanol；
Type of elution：Gradient elution；
Gradient elution program is：
0-5min：Supercritical CO2Volume fraction be down to 85% by 95%,
5.0-5.5min：Supercritical CO2Volume fraction be down to 80% by 85%,
5.5-6.0min：Supercritical CO2Volume fraction increase to 95% by 80%,
6.0-7.0min：Supercritical CO2Volume fraction remain 95% constant；
Column flow rate is 1.55-1.65mL/min；
The detector that the efficiently conjunction phase chromatography uses is PDA detector；
Wherein, 12 kinds of sulfa drugs are selected from following substances：Sulfamethoxazole, madribon, sulfanilamide (SN) Vinegar acyl, sulfamethyldiazine, sulphadiazine, sulphathiazole, sulfapryidine, N4- sulfacetamide methyl oxazole, N4- sulfacetamide first Yl pyrimidines, N4- acetylsulfadiazine, N4- acetylsulfathiazole, N4- acetylsulfapyridine.
2. according to the method described in claim 1, it is characterized in that：It is described to mix by the way of being vortexed in the step 1), The time of vortex is 0.5-1min；The condition of the centrifugation is that 6000~10000rpm centrifuges 6~12min at 18~24 DEG C.
3. method according to claim 1 or 2, it is characterised in that：In the step 2), the Acquity UPC2BEH colors The specification of spectrum column is 100mm × 3mm, 1.7 μm of packing material size；The column temperature is：40℃；The system back pressure：2100psi；
Sample temperature is 25 DEG C in the chromatographic condition, and sampling volume is 2 μ L.
4. method according to any one of claim 1-3, it is characterised in that：In the step 2), the PDA detectors Testing conditions be：
3D scanning ranges 210-600nm, 2D compensation absorbance 265nm, compensation reference 560-600nm.
5. method that is a kind of while measuring the content of 12 kinds of sulfa drugs in blood plasma, is surveyed using efficiently conjunction phase chromatography It is fixed, comprise the following steps：
1) preparation of standard curve
The hybrid standard product solution of 12 kinds of sulfa drugs described in a series of claim 1 of concentration is taken to add blank plasma sample In product, plasma sample is handled according to the method described in step 1) in claim 1, then to obtained supernatant according to The efficient phase chromatography that closes described in claim 1 is detected, and records 12 kinds of sulfa drugs respectively in each concentration Under corresponding peak area,
Using the corresponding peak area of each sulfa drugs as ordinate, using the mass concentration of corresponding sulfa drugs as horizontal seat Mark, carries out linear weight recurrence, prepares the regression equation of 12 kinds of sulfa drugs respectively；
2) assay of 12 kinds of sulfa drugs described in plasma sample to be measured
Test plasma sample is handled according to the method described in step 1) in claim 1, then the supernatant to obtaining The phase chromatography described in accordance with the claim 1 that efficiently closes is detected, and it is corresponding to record 12 kinds of sulfa drugs respectively Peak area；The peak area value of every kind of medicine is substituted into the corresponding regression equation of the medicine prepared by step 1), calculated respectively The concentration of 12 kinds of sulfa drugs into the test plasma sample.
6. according to the method described in claim 5, it is characterized in that：It is described to mix by the way of being vortexed in the step 1), The time of vortex is 0.5-1min；The condition of the centrifugation is that 6000~10000rpm centrifuges 6~12min at 18~24 DEG C.
7. the method according to claim 5 or 6, it is characterised in that：In the step 2), the Acquity UPC2BEH colors The specification of spectrum column is 100mm × 3mm, 1.7 μm of packing material size；The column temperature is：40℃；The system back pressure：2100psi；
Sample temperature is 25 DEG C in the chromatographic condition, and sampling volume is 2 μ L.
8. according to the method any one of claim 5-7, it is characterised in that：In the step 2), the PDA detectors Testing conditions be：
3D scanning ranges 210-600nm, 2D compensation absorbance 265nm, compensation reference 560-600nm.
9. application of the method in sulfa drugs pharmacokinetics is studied any one of claim 1-8；The sulphur Amine drug is selected from following at least one：Sulfamethoxazole, madribon, sulfacetamide, methylene sulfonamide are phonetic Pyridine, sulphadiazine, sulphathiazole, sulfapryidine, N4- sulfacetamide methyl oxazole, N4- sulfacetamide methylpyrimidine, N4- acetyl Sulphadiazine, N4- acetylsulfathiazole, N4- acetylsulfapyridine.
CN201711212283.6A 2017-11-28 2017-11-28 Method for simultaneously measuring 12 sulfonamides in blood plasma by adopting high-efficiency synthetic phase chromatography Expired - Fee Related CN107991409B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111189938A (en) * 2020-01-10 2020-05-22 华中农业大学 Quantitative detection method for adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004098600A1 (en) * 2003-05-06 2004-11-18 Astrazeneca Ab Positive modulators of nicotinic acetylcholine receptors
CN107531705A (en) * 2015-03-02 2018-01-02 美国安进公司 Bicyclic one sulfonamide compound

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004098600A1 (en) * 2003-05-06 2004-11-18 Astrazeneca Ab Positive modulators of nicotinic acetylcholine receptors
CN107531705A (en) * 2015-03-02 2018-01-02 美国安进公司 Bicyclic one sulfonamide compound

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SUVARNA I. BHOIR等: "Determination of sulfadoxine in human blood plasma using packed-column supercritical fluid chromatography", 《JOURNAL OF CHROMATOGRAPHY B》 *
ZHANG YUAN等: "A simple, accurate, time-saving and green method for the determination of 15 sulfonamides and metabolites in serum samples by ultra-high performance supercritical fluid chromatography", 《JOURNAL OF CHROMATOGRAPHY A》 *
张元等: "食品中磺胺类药物前处理及检测方法研究进展", 《食品科学》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111189938A (en) * 2020-01-10 2020-05-22 华中农业大学 Quantitative detection method for adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid

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